IL257500B2 - Modified cullin1 gene - Google Patents

Modified cullin1 gene

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Publication number
IL257500B2
IL257500B2 IL257500A IL25750018A IL257500B2 IL 257500 B2 IL257500 B2 IL 257500B2 IL 257500 A IL257500 A IL 257500A IL 25750018 A IL25750018 A IL 25750018A IL 257500 B2 IL257500 B2 IL 257500B2
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seq
plant
modified
gene
cullinl
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IL257500A
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Rijk Zwaan Zaadteelt En Zaadhandel Bv
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Publication of IL257500B2 publication Critical patent/IL257500B2/en

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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00Processes for modifying genotypes ; Plants characterised by associated natural traits
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01GHORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00Cultivation of specific crops or plants not otherwise provided for
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01GHORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00Cultivation of specific crops or plants not otherwise provided for
    • A01G22/15Leaf crops, e.g. lettuce or spinach 
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01GHORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00Cultivation of specific crops or plants not otherwise provided for
    • A01G22/35Bulbs; Alliums, e.g. onions or leeks
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146Genetically Modified [GMO] plants, e.g. transgenic plants

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Description

PCT/EP2016/071177 WO 2017/042270 MODIFIED CULLIN1 GENE The present invention relates to a plant type that enables efficient cultivation, and to methods for identifying and developing such a plant. The present invention also relates to a plant type suitable for situations that require smaller portions, or reduces the labour, time and expenses involved with storing, handling and transporting of redundant plant leaves, and to methods for identifying and developing such plant.Plant breeders are continuously looking for more efficient ways to cultivate plants. One way to increase the efficiency is by high-wire cultivation. In the high-wire cultivation, higher planting densities are used to obtain higher yields per m2. In addition, high-wire cultivation allows a longer cultivation period during which the plant produces fruits. Cucumber and tomato are crops that are suitable for high-wire cultivation. However, not all varieties are fit for this type of cultivation.More efficient cultivation can also be achieved in other crops if the plants have a compact growth phenotype. Such a compact growth phenotype may be characterized by the plants showing shorter internodes and/or a smaller leave area. Because of this compact growth phenotype, the plants can be planted in higher densities, thereby saving a lot of space.It is one goal of the present invention to provide a plant type that enables efficient cultivation. This goal has been achieved by providing a plant type showing a compact growth phenotype.In the research leading to the present invention, it was found that a mutation in the Cullinl gene leads to a modification in plant type that may be expressed as a compact growth phenotype. A plant that has the mutant gene is in particular suitable for efficient cultivation. Such a plant shows a shorter internode length and/or a smaller leaf area and may also display other characteristics that lead to a compact growth phenotype.Cullin proteins are a family of proteins present in all eukaryotes, not only plants. The Cullin proteins combine with RING proteins to form so-called Cullin-RING ubiquitine ligases (CRLs). In general, Cullin proteins play an important role in protein ubiquitination and protein degradation.Ubiquitination (also known as ubiquitylation) is an enzymatic, post-translational modification process in which an ubiquitin protein is ligated to substrate protein.Ubiquitine is a highly conserved, small polypeptide, ubiquitously distributed among eukaryotes. An ATP-dependent reaction cascade (involving the sequential action of ubiquitine- activating (Els), ubiquitine-conjugating (E2s) and ubiquitine-protein ligase (E3s) enzymes) performs the ligation of ubiquitine to other proteins. Proteins that are ligated with ubiquitine are subsequently degraded and broken down, or relocated.
PCT/EP2016/071177 WO 2017/042270 The Cullinl protein, which is a member of the Cullin protein family, is one out of the four subunits that make up the SCF complex. The abbreviation SCF stands for SKPl-CULl-F-box protein E3 ubiquitin ligase complex, which mediates the ubiquitination of proteins involved in cell cycle progression, signal transduction and transcription. In the SCF complex, Cullinl serves as a rigid scaffold that organizes the SKPl-F-box protein and RBX1 subunits. It may contribute to catalysis through positioning of the substrate and the ubiquitin-conjugating enzyme.As mentioned before, Cullin proteins are present in all eukaryotes. Through its involvement in ubiquitination and subsequent processes it is engaged in a wide range of cellular processes. Surprisingly, the mutation of the invention causes shorter internode length and/or smaller leaf area without causing a deleterious effect on the plant, which would be expected based on the state of the art knowledge available on the conserved status of Cullinl protein and its functionality.The characterisation of the modified Cullinl gene in the present research was performed in cucumber (Cucumis sativus). This enabled the identification of further crops having a Cullinl gene, which when modified leads to plants with a compact growth phenotype, i.e. showing shorter internode length and/or a smaller leaf area, or a reduction in other plant parts when compared to plants not having the modified Cullinl gene. These crops include those belonging to the family of Cucurbitacea, such as for instance melon (Cucumis melo), watermelon (Citrullus lanatus), and squash (Cucurbita pepo)׳ , fruit crops, such as pepper (Capsicum annuum), tomato (Solanum lycopersicum), and eggplant (Solanum melongena)׳ , leafy vegetables, such as lettuce (Lactuca sativa), spinach (Spinacia oleracea), chicory (Cichorium intybus), and cabbage (Brassica oleracea)׳ , root vegetables, such as for instance carrot (Daucus carota), radish (Raphanus sativus), and beetroot (Beta vulgaris)׳ , and other crops, such as celery (Apium graveolens) and leek (.Allium ampeloprasum).It is another goal of the present invention to provide a plant type that is suitable for situations that require smaller portions, or reduces the labour, time and expenses involved with storing, handling and transporting of redundant plant leaves.The invention thus relates to a modified Cullinl gene comprising a modification in the wild type Cullinl nucleotide sequence which leads to a modification in the wild type Cullinl amino acid sequence.The Cullinl gene can be modified by different means known in the art, including mutagenesis. Mutagenesis comprises the random introduction of at least one modification to DNA by means of one or more chemical compounds, such as ethyl methanesulphonate (EMS), nitrosomethylurea, hydroxylamine, proflavine, N-methyl-N-nitrosoguanidine, N-ethyl-N- nitrosourea, N-methyl-N-nitro-nitrosoguanidine, diethyl sulphate, ethylene inline, sodium azide, formaline, urethane, phenol and ethylene oxide, and/or by physical means, such as UV-irradiation, PCT/EP2016/071177 WO 2017/042270 fast-neutron exposure, X-rays, gamma irradiation, and/or by insertion of genetic elements, such as transposons, T-DNA, retroviral elements. Mutagenesis also comprises the more specific, targeted introduction of at least one modification by means of homologous recombination, oligonucleotide- based mutation induction, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems.The modified Cullinl gene may be an exogenous Cullinl gene introduced into a plant by a transgenic method or a cisgenic method. Use of a modified Cullinl gene of the invention for developing a plant that shows a compact growth phenotype, i.e. comprises shorter internodes and/or smaller leaf area, comprises the introduction of a modified exogenous Cullinl gene by a transgenic or a cisgenic method.The modified Cullinl gene may be part of a gene construct, which gene construct comprises a selectable marker, a promoter sequence, a Cullinl gene sequence, and a terminator sequence.The present invention is widely applicable to all plant species that have a functional orthologue of the Cullinl gene in their genome, i.e. an orthologue that performs the same or a similar biological function. Identification of Cullinl orthologues, i.e. Cullinl genes in other species, can be performed in many crops, methods of which are known in the art. The present invention can for instance be applied to a plant belonging to a species selected from the group consisting of Cucumis sativus, Cucumis melo, Cucurbita pepo, Citrullus lanatus, Solanum melongena, Solanum lycopersicum, Capsicum annuum, Brassica oleracea, Daucus carota, Apium graveolens, Cichorium intybus, Cichorium endivia, Allium ampeloprasum, Lactuca sativa, Raphanus sativus, Spinacia oleracea, and Beta vulgaris.Accordingly, the present invention relates to a modified Cullinl gene comprising a modification in the wild type Cullinl nucleotide sequence of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 which leads to a modification in the wild type Cullinl amino acid sequence of SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No:27, SEQ ID No: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO. 33, SEQ ID NO: 34 respectively.Figures 1-17 show the wild type Cullinl nucleotide sequences SEQ ID NO. 1-17 of Cucumis sativus, Cucumis melo, Cucurbita pepo, Citrullus lanatus, Solanum melongena, Solanum lycopersicum, Capsicum annuum, Brassica oleracea, Daucus carota, Apium graveolens,Cichorium intybus, Cichorium endivia, Allium ampeloprasum, Lactuca sativa, Raphanus sativus, PCT/EP2016/071177 WO 2017/042270 Spinacia oleracea, and Beta vulgaris, respectively. Figures 18-34 show the wild type Cullinl amino acid sequences SEQ ID NO. 18-34 of Cucumis sativus, Cucumis melo, Cucurbita pepo, Citrullus lanatus, Solanum melongena, Solanum lycopersicum, Capsicum annuum, Brassica oleracea, Daucus carota, Apium graveolens, Cichorium intybus, Cichorium endivia, Allium ampeloprasum, Lactuca sativa, Raphanus sativus, Spinacia oleracea, and Beta vulgaris, respectively.The term "wild type" as used herein refers in general to the form of an organism, gene, protein, or trait as it would occur in nature, as opposed to a mutated or modified form. In this application wild type refers specifically to the naturally occurring form of the Cullinl gene, the naturally occurring form of the nucleotide sequence of Cullinl and the naturally occurring form of the Cullinl amino acid sequence. The naturally occurring forms of the Cullinl gene and Cullinl protein of several crops are shown in Figures 1-17 and Figures 18 -34 respectively.The terms "mutant", "mutation", "modification" ,"modified", "mutated Cullinl gene" and "modified Cullinl gene" as used herein refer to nucleotide changes and amino acid changes to the wild type Cullinl gene thereof that lead to a modified version of the wild type gene. The modification can be any modification, including but not limited to a SNP.The modified Cullinl gene is also referred to herein as "the gene of the invention", "the modified Cullinl gene", or "the modified Cullinl gene of the invention". These terms are used interchangeably herein. As used herein the phrase "the modified Cullinl gene" is intended to encompass the Cullinl gene with any modification that leads to the compact growth phenotype.The terms "compact gene phenotype", "compact phenotype" or "compact growth phenotype" are used interchangeably herein and refer to a phenotype of a shorter internode length and/or a smaller leaf area. Crops comprising the modified Cullinl gene and which have internodes, such as for instance cucumber, may show shorter internodes or a smaller leaf area. They may also show shorter internodes and a smaller leaf area. Crops comprising the modified Cullinl gene but which do not have internodes, such as for instance lettuce, may show a smaller leaf area.The term "smaller leaf area" as used herein is the leaf area that displays a reduction in individual leaf area of, in order of increased preference, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% as a result of the homozygous or heterozygous presence of the modified gene of the invention. To investigate the influence of the gene of the invention on the smaller leaf area, a skilled person would have to compare plants having the gene of the invention homozygously or heterozygously with plants that are isogenic to first mentioned plants but do not have the gene of the invention.With the term "leaf’ is meant the part of the plant consisting of the petiole and leaf blade. With the term "leaf area" is meant the surface of the part of the plant consisting of the leaf blade.
PCT/EP2016/071177 WO 2017/042270 The term "shorter internodes" or "shorter internode length" as used herein is internode length that has a reduction in individual length of, in order of increased preference, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% as a result of the homozygous or heterozygous presence of the gene of the invention. To investigate the influence of the gene of the invention on the shorter internode length, a skilled person would have to compare plants having the gene of the invention homozygously or heterozygously with plants that are isogenic to first mentioned plants but without the gene of the invention.The modification leading to the modified Cullinl gene may be selected from a modification that changes the mRNA level of the Cullinl gene, a modification that changes the Cullinl protein structure and/or levels, and/or a modification that changes the Cullinl protein activity.One aspect of the invention relates to a modified Cullinl gene, comprising a mutation as compared to its wild type genomic sequence, which mutation leads to a change in the Cullinl protein and/or protein activity, wherein the modified Cullinl gene is capable of causing a compact growth phenotype.In one embodiment, the mutation is a Single Nucleotide Polymorphism (SNP).In one embodiment of the present invention, the change in the amino acid sequence is a substitution.In a preferred embodiment of the present invention, the change in the amino acid sequence is found in the part of the Cullinl protein between the amino acids on position 30-60, preferably in the part between the amino acids on positions 40-55 of the cucumber amino acid sequence of SEQ ID No: 18, or on a part corresponding thereto.In a further preferred embodiment of the present invention, the change in the amino acid sequence is found in the part of the Cullinl protein that binds SKP1 and/or ETA2.Preferably, the physical location of the amino acid substitution in the Cullinl protein lies in the region of the protein where the Cullinl protein binds to SKP1 and/or ETA2. The Cullinl protein forms a so-called SCF complex, together with SKP1, RBX1 and a F-box protein, which has important functionalities such as a role in leaf development. ETA2 is according to some theories, required to sustain the SCF complex activity, most probably by facilitating cycles of assembly and disassembly of the SCF complex.In a specific embodiment, the modified Cullinl gene includes, a Cullinl gene comprising a SNP on position 147 of SEQ ID NO.l of cucumber, or on a position corresponding thereto in the Cullinl gene of other crops, wherein the modification comprises a change in the nucleotide on that position and wherein the modification leads to an amino acid substitution in the Cullinl protein on position 49 of the wildtype protein sequence SEQ ID NO: 18. of cucumber, or on a position PCT/EP2016/071177 WO 2017/042270 corresponding thereto, in other crops. In cucumber the change is from A to G and from Isoleucine to Methionine. In other crops the change in nucleotide and amino acid may be different.In a preferred embodiment of the present invention, the modification of the nucleotide sequence comprises a change from Adenine, Cytosine or Thymine to Guanine.The definition "coding sequence" as used herein is the portion of the gene’s DNA composed of exons that code for the protein.In a further embodiment of the present invention, the modification in the amino acid sequence is a substitution on position 49 of the cucumber amino acid sequence of SEQ ID No: 18, or, in case of a crop other than cucumber, on a position corresponding to position 49 of the cucumber amino acid sequence of SEQ ID No: 18.The amino acid substitution caused by the mutation of the current invention was found to be present on position 49 of the cucumber amino acid sequence of SEQ ID No: 18, or, in case of a crop other than cucumber, on a position corresponding to position 49 of the wild type amino acid sequence SEQ ID NO. 18 of cucumber. This nucleotide mutation is considered to be non- conservative, and the amino acid change can be considered non-conservative.Amino acid changes in a protein occur when the mutation of one or more base pairs in the coding DNA sequence result in an altered codon triplet that encodes a different amino acid. Not all point mutations in the coding DNA sequence lead to amino acid changes, due to the redundancy of the genetic code. Mutations in the coding sequence that do not lead to amino acid changes are called "silent mutations". Other mutations are called "conservative", they lead to the replacement of one amino acid by another amino acid with comparable properties, such that the mutations are unlikely to change the folding of the mature protein, or influence its function. As used herein a "non-conservative amino acid change" refers to an amino acid that is replaced by another amino acid that has different chemical properties that may lead to decreased stability, changed functionality and/or structural effects of the encoded protein.In a further preferred embodiment of the present invention, the modification in the amino acid sequence is a substitution and consists of a change from Isoleucine to MethionineThe invention further relates to a plant comprising the modified Cullinl gene.A Cucumis sativus plant comprising the modified Cullinl gene with the nucleotide sequence of SEQ ID No: 35 is not part of this invention and is therefore disclaimed herewith.A plant comprising the modified Cullinl gene shows a compact growth phenotype, i.e. comprises a shorter internode length and/or a smaller leaf area, compared to an isogenic plant of the same species not comprising the modified Cullinl gene. For example, a Cucumis sativus plant a Cucumis melo plant, a Cucurbita pepo plant, a Citrullus lanatus plant, a Solanum melongena plant, a, Solanum lycopersicum plant, and a Capsicum annuum plant comprising the modified Cullinl gene show a shorter internode length and/or a smaller leaf area. These plants are therefore PCT/EP2016/071177 WO 2017/042270 particularly suitable for efficient cultivation. A Cichorium intybus plant, a Cichorium endivia plant, a Lactuca sativa plant, a Brassica oleracea plant and a Spinacia oleracea plant comprising the modified Cullinl gene show for example a smaller leaf area. As such, the parts of these plants harvested for consumption are smaller in size. These plants are therefore particularly suitable for a situation in which smaller portions are required. A Daucus carota plant comprising the modified Cullinl gene shows a shorter internode length and/or a smaller leaf area. As such, the plant comprises less foliage that needs to be removed before the product is sold and/or consumed. An Apium graveolens plant comprising the modified Cullinl gene shows a shorter internode length and/or a smaller leaf area. As such, the plant is of a more compact size suitable for a situation in which smaller portions are required and/or comprises less foliage that needs to be removed before the product is sold and/or consumed. An Allium ampeloprasum plant comprising the modified Cullinl gene shows a smaller leaf area. As such, the leaves that are not consumed do not need to be removed before the product is sold and/or consumed. A Raphanus sativus plant and a Beta vulgaris plant comprising the modified Cullinl gene show a smaller leaf area. As such, less foliage needs to be removed before the product is sold and/or consumed.The plant of the invention comprising the modified Cullinl gene either homozygously or heterozygously may be a plant of an inbred line, a hybrid, a doubled haploid, or a plant of a segregating population.The plant of the invention may have the modified Cullinl gene in heterozygous state since such a plant shows the trait in an intermediate level. Furthermore such plant may be a potential source of the gene and when crossed with another plant that optionally also has the modified gene either homozygously or heterozygously can result in progeny plants that have the modified gene homozygously or heterozygously and show the trait of having a compact growth phenotype.The invention also relates to a method for the production of a plant having the modified Cullinl gene that leads to a compact growth phenotype by using a seed that comprises the modified Cullinl gene for growing the said plant.The invention further relates to a method for the production of a plant having the modified Cullinl gene by using tissue culture of plant material that carries the modified Cullinl gene in its genome.The invention furthermore relates to a method for the production of a plant having the modified Cullinl gene which leads to a compact growth phenotype, by using vegetative reproduction of plant material that carries the modified Cullinl gene in its genome.The invention further provides a method for the production of a plant having the modified Cullinl gene by using a doubled haploid generation technique to generate a doubled haploid line from a cucumber plant comprising the modified Cullinl gene.
PCT/EP2016/071177 WO 2017/042270 The invention further relates to a plant seed comprising the modified Cullinl gene of the invention, wherein the plant that can be grown from the seed shows a compact growth phenotype.The invention also relates to a method for seed production comprising growing plants from seeds of the invention, allowing the plants to produce seeds by allowing pollination to occur, and harvesting those seeds. Production of the seeds is suitably done by crossing or selfing. Seeds produced in that manner result in a compact growth phenotype of the plants grown thereof.The invention furthermore relates to hybrid seed and to a method for producing such hybrid seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, wherein said first parent plant and/or said second parent plant has the modified Cullinl gene of the invention. A hybrid plant resulting from growing the resulting seed that comprises the modified Cullinl gene of the invention, showing the compact growth phenotype of the invention is also a plant of the invention.Another aspect of the invention relates to propagation material capable of developing into and/or being derived from a plant comprising a modified Cullinl gene, wherein the plant shows a compact growth phenotype, compared to an isogenic plant of the same species not comprising the modified Cullinl gene, wherein the propagation material comprises the modified Cullinl gene of the invention and wherein the propagation material is selected from the group consisting of microspores, pollen, ovaries, ovules, embryos, embryo sacs, egg cells, cuttings, roots, hypocotyls, cotyledons, stems, leaves, flowers, anthers, seeds, meristematic cells, protoplasts and cells, or tissue culture thereof.The invention thus further relates to parts of a claimed plant that are suitable for sexual reproduction. Such parts are for example selected from the group consisting of microspores, pollen, ovaries, ovules, embryo sacs, and egg cells. In addition, the invention relates to parts of a claimed plant that are suitable for vegetative reproduction, which are in particular cuttings, roots, stems, cells, protoplasts. The parts of the plants as mentioned above are considered propagation material. The plant that is produced from the propagation material comprises the modified Cullinl gene that leads to a compact growth phenotype and thus enables efficient cultivation and/or is suitable for the production for market segments that require a smaller product.According to a further aspect thereof, the invention provides a tissue culture of a plant carrying the modified Cullinl gene of the invention, which is also propagation material. The tissue culture comprises regenerable cells. Such tissue culture can be selected or derived from any part of the plant, in particular from leaves, pollen, embryos, cotyledon, hypocotyls, meristematic cells, roots, root tips, anthers, flowers, seeds, and stems. The tissue culture can be regenerated into a plant carrying the modified Cullinl gene of the invention, which regenerated plant expresses the trait of the invention and is also part of the invention.
PCT/EP2016/071177 WO 2017/042270 The invention further relates to the use of the modified Cullinl gene for the development of a plant showing a compact growth phenotype. A skilled person is familiar with introducing a new trait into a plant already having other desired agricultural properties, for instance by introgression. Introgression can be done by means of standard breeding techniques, wherein selection can be done either phenotypically or with the use of markers or a combination thereof.The invention further relates to the use of the modified Cullinl gene, or a part thereof comprising the modification, as a marker for identifying a plant showing a compact growth phenotype.The ‘use for identifying’ or a ‘method for identifying’ as used in the current application comprises the use of the described (causal) SNP in the Cullinl gene as a marker. The invention also relates to other markers that can be developed based on a modification, including the causal SNP, in the Cullinl gene, as well as to other markers that can be developed based on the wildtype sequence of the Cullinl gene.The invention further relates to the use of any of the sequences of SEQ ID NOs. 35-51, or a part thereof, as a marker for identifying a plant showing a compact growth phenotype, i.e. comprising a shorter internode length and/or a smaller leaf area. If a part of any of these sequences is used, the part must comprise the modification. For example, SEQ ID No. 35 or a part thereof may be used to identify a Cucumis sativus plant showing a shorter internode length and/or a smaller leaf area; SEQ ID No. 36 or a part thereof may be used to identify a Cucumis melo plant showing a shorter internode length and/or a smaller leaf area; SEQ ID No. 37 or a part thereof may be used to identify a Cucurbita pepo plant showing a shorter internode length and/or a smaller leaf area; SEQ ID No. 38 or a part thereof may be used to identify a Citrullus lanatus plant showing a shorter internode length and/or a smaller leaf area; SEQ ID No. 39 or a part thereof may be used to identify a Solanum melongena plant showing a shorter internode length and/or a smaller leaf area; SEQ ID No. 40 or a part thereof may be used to identify a Solanum lycopersicum plant showing a shorter internode length and/or a smaller leaf area; SEQ ID No. 41 or a part thereof may be used to identify a Capsicum annuum plant showing a shorter internode length and/or a smaller leaf area; SEQ ID No. 42 or a part thereof may be used to identify a Brassica oleracea plant showing a smaller leaf area; SEQ ID No. 43 or a part thereof may be used to identify a Daucus carota plant showing a shorter internode length and/or a smaller leaf area; SEQ ID No. 44 or a part thereof may be used to identify a Apium graveolens plant showing a shorter internode length and/or a smaller leaf area; SEQ ID No. 45 or a part thereof may be used to identify a Cichorium intybus plant showing a smaller leaf area; SEQ ID No. 46 or a part thereof may be used to identify a Cichorium endivia plant showing a smaller leaf area; SEQ ID No. 47 or a part thereof may be used to identify an Allium ampeloprasum plant showing a smaller leaf area; SEQ ID No. 48 or a part thereof may be used to identify a Lactuca sativa plant showing a smaller leaf area; SEQ ID No. 49 or a part PCT/EP2016/071177 WO 2017/042270 thereof may be used to identify a Raphanus sativus plant showing a shorter internode length and/or a smaller leaf area; SEQ ID No. 50 or a part thereof may be used to identify a Spinacia oleracea plant showing a smaller leaf area; SEQ ID No. 51 or a part thereof may be used to identify a Beta vulgaris plant showing a shorter internode length and/or a smaller leaf area. The invention also relates to the use of any markers that are derived from SEQ ID Nos. 1-17 or SEQ ID NOs. 36-for identifying a plant showing a compact growth phenotype. Any such derived marker must comprise the modification that leads to the phenotype of the invention.In general, to identify a plant showing a compact growth phenotype, it is thus determined in the Cullinl gene whether there is an A, C, or T (SEQ ID Nos. 1-17) or a G (SEQ ID Nos. 35-51) on position 147, or a position corresponding thereto.The invention further relates to a method for obtaining a plant which shows a compact growth phenotype comprising;a) crossing a plant comprising the modified Cullinl gene of the current invention with a plant not comprising the modified Cullinl gene, to obtain an FI population;b) optionally performing one or more rounds of selfing and/or crossing a plant from the FI to obtain a further generation population; andc) selecting a plant that has a compact growth phenotype and the modified Cullinl gene of the invention.The invention also relates to a method for obtaining a plant which shows a compact growth phenotype comprising;a) crossing a plant comprising the modified Cullinl gene of the current invention with another plant comprising the modified Cullinl gene, to obtain an FI population;b) optionally performing one or more rounds of selfing and/or crossing a plant from the FI to obtain a further generation population; andc) selecting a plant that has a compact growth phenotype and the modified Cullinl gene of the invention.The invention further relates to a marker for identifying a plant showing a compact growth phenotype, comprising the modified Cullinl, or a part thereof that comprises the modification. Preferably, the modification is a nucleotide substitution on or around position 147 of SEQ ID No:l of cucumber, or, in case of a crop other than cucumber, on or around a position corresponding to position 147 of SEQ ID No:l of cucumber, which modification leads to an amino acid substitution in the Cullinl protein.The invention also relates to a method for selecting a plant showing a compact growth phenotype from a population of plants, comprising detecting the presence or absence of a guanine on position 147 of the cucumber nucleotide sequence of SEQ ID NO:l, or, for a crop other than cucumber, on a position corresponding to position 147 of the cucumber nucleotide PCT/EP2016/071177 WO 2017/042270 sequence of SEQ IDNo: 1, in the genome of a plant of a population of plants, and selecting a plant comprising a guanine on position 147 of SEQ IDNO:l, as shown in SEQ IDNO. 35, or, for a crop other than cucumber, on a position corresponding to position 147 of the cucumber nucleotide sequence of SEQ IDNo: 1, as shown in any of SEQ IDNos. 36- 51.
FIGURES Figure 1Cucumber Cullinl coding sequence wildtype, SEQ IDNO. 1.The nucleotide between brackets and bold indicates the position of the SNP, 147 nucleotides from the start. The wildtype nucleotide is "A", as shown here. Figure 2Melon Cullinl coding sequence wildtype, SEQ IDNO. 2. The nucleotide between brackets and bold indicates the position of the SNP, 147 nucleotides from the start. The wildtype nucleotide is "A", as shown here. Figure 3Squash Cullinl coding sequence wildtype, SEQ IDNO. 3. The nucleotide between brackets and bold indicates the position of the SNP, 147 nucleotides from the start. The wildtype nucleotide is "A", as shown here. Figure 4Watermelon Cullinl coding sequence wildtype, SEQ IDNO. 4. The nucleotide between brackets and bold indicates the position of the SNP, 147 nucleotides from the start. The wildtype nucleotide is "A", as shown here. Figure 5Eggplant Cullinl coding sequence wildtype, SEQ IDNO. 5. The nucleotide between brackets and bold indicates the position of the SNP, 141 nucleotides from the start. The wildtype nucleotide is "T", as shown here. Figure 6Tomato Cullinl coding sequence wildtype, SEQ IDNO. 6. The nucleotide between brackets and bold indicates the position of the SNP, 141 nucleotides from the start. The wildtype nucleotide is "T", as shown here. Figure 7Pepper Cullinl coding sequence wildtype, SEQ IDNO. 7. The nucleotide between brackets and bold indicates the position of the SNP, 141 nucleotides from the start. The wildtype nucleotide is "T", as shown here. Figure 8Cabbage Cullinl coding sequence wildtype, SEQ IDNO. 8. The nucleotide between brackets and bold indicates the position of the SNP, 138 nucleotides from the start. The wildtype nucleotide is "C", as shown here. Figure 9Carrot Cullinl coding sequence wildtype, SEQ IDNO. 9. The nucleotide between brackets and bold indicates the position of the SNP, 144 nucleotides from the start. The wildtype nucleotide is "C", as shown here. Figure 10Celery Cullinl coding sequence wildtype, SEQ IDNO. 10. The nucleotide between brackets and bold indicates the position of the SNP, 141 nucleotides from the start. The wildtype nucleotide is "C", as shown here.
PCT/EP2016/071177 WO 2017/042270 Figure 11Chicory Cullinl coding sequence wildtype, SEQ IDNO. 11.The nucleotide between brackets and bold indicates the position of the SNP, 141 nucleotides from the start. The wildtype nucleotide is "C", as shown here. Figure 12Endive Cullinl coding sequence wildtype, SEQ IDNO. 12. The nucleotide between brackets and bold indicates the position of the SNP, 141 nucleotides from the start. The wildtype nucleotide is "C", as shown here. Figure 13Leek Cullinl coding sequence wildtype, SEQ IDNO. 13. The nucleotide between brackets and bold indicates the position of the SNP, 147 nucleotides from the start. The wildtype nucleotide is "C", as shown here. Figure 14Lettuce Cullinl coding sequence wildtype, SEQ IDNO. 14. The nucleotide between brackets and bold indicates the position of the SNP, 141 nucleotides from the start. The wildtype nucleotide is "C", as shown here. Figure 15Radish Cullinl coding sequence wildtype, SEQ IDNO. 15. The nucleotide between brackets and bold indicates the position of the SNP, 138 nucleotides from the start. The wildtype nucleotide is "C", as shown here. Figure 16Spinach Cullinl coding sequence wildtype, SEQ IDNO. 16. The nucleotide between brackets and bold indicates the position of the SNP, 141 nucleotides from the start. The wildtype nucleotide is "A", as shown here. Figure 17Beetroot Cullinl coding sequence wildtype, SEQ IDNO. 17. The nucleotide between brackets and bold indicates the position of the SNP, 141 nucleotides from the start. The wildtype nucleotide is "A", as shown here. Figure 18Cucumber Cullinl protein sequence wildtype, SEQ IDNO. 18. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 19Melon Cullinl protein sequence wildtype, SEQ IDNO. 19. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 20Squash Cullinl protein sequence wildtype, SEQ IDNO.20. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 21Watermelon Cullinl protein sequence wildtype, SEQ IDNO.21. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 22Eggplant Cullinl protein sequence wildtype, SEQ IDN0.22. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here.
Figure 23Tomato Cullinl protein sequence wildtype, SEQ IDN0.23. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 24Pepper Cullinl protein sequence wildtype, SEQ IDN0.24. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 25Cabbage Cullinl protein sequence wildtype, SEQ IDN0.25. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 26Carrot Cullinl protein sequence wildtype, SEQ IDN0.26. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 27Celery Cullinl protein sequence wildtype, SEQ IDN0.27. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 28Chicory Cullinl protein sequence wildtype, SEQ IDN0.28. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 29Endive Cullinl protein sequence wildtype, SEQ IDN0.29. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 30Leek Cullinl protein sequence wildtype, SEQ IDNO.30. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure31 Lettuce Cullinl protein sequence wildtype, SEQ IDNO.31. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 32Radish Cullinl protein sequence wildtype, SEQ IDN0.32. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 33Spinach Cullinl protein sequence wildtype, SEQ IDN0.33. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here. Figure 34Beetroot Cullinl protein sequence wildtype, SEQ IDNO.34. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The wildtype amino acid is ‘I’,as shown here.
WO 2017/042270 PCT/EP2016/071177 13 PCT/EP2016/071177 WO 2017/042270 Figure 35Cucumber Cullinl coding sequence "modified" SEQ IDNO. 35. The nucleotide between brackets and bold and bold indicates the position of the SNP 147 bp from the start. The modified nucleotide is "G", as shown here. Figure 36Melon Cullinl coding sequence "modified" SEQ IDNO. 36. The nucleotide between brackets and bold indicates the position of the SNP 147 bp from the start. The modified nucleotide is "G", as shown here. Figure 37Squash Cullinl coding sequence "modified" SEQ IDNO. 37. The nucleotide between brackets and bold indicates the position of the SNP 147 bp from the start. The modified nucleotide is "G", as shown here. Figure 38Watermelon Cullinl coding sequence "modified" SEQ IDNO. 38. The nucleotide between brackets and bold indicates the position of the SNP 147 bp from the start. The modified nucleotide is "G", as shown here. Figure 39Eggplant Cullinl coding sequence "modified" SEQ IDNO. 39. The nucleotide between brackets and bold indicates the position of the SNP 141 bp from the start. The modified nucleotide is "G", as shown here. Figure 40Tomato Cullinl coding sequence "modified" SEQ IDNO. 40. The nucleotide between brackets and bold indicates the position of the SNP 141 bp from the start. The modified nucleotide is "G", as shown here. Figure 41Pepper Cullinl coding sequence "modified" SEQ IDNO. 41. The nucleotide between brackets and bold indicates the position of the SNP 141 bp from the start. The modified nucleotide is "G", as shown here. Figure 42Cabbage Cullinl coding sequence "modified" SEQ IDNO. 42. The nucleotide between brackets and bold indicates the position of the SNP 138 bp from the start. The modified nucleotide is "G", as shown here. Figure 43Carrot Cullinl coding sequence "modified" SEQ IDNO. 43. The nucleotide between brackets and bold indicates the position of the SNP 144 bp from the start. The modified nucleotide is "G", as shown here. Figure 44Celery Cullinl coding sequence "modified" SEQ IDNO. 44. The nucleotide between brackets and bold indicates the position of the SNP 141 bp from the start. The modified nucleotide is "G", as shown here. Figure 45Chicory Cullinl coding sequence "modified" SEQ IDNO. 45. The nucleotide between brackets and bold indicates the position of the SNP 141 bp from the start. The modified nucleotide is "G", as shown here. Figure 46Endive Cullinl coding sequence "modified" SEQ IDNO. 46. The nucleotide between brackets and bold indicates the position of the SNP 141 bp from the start. The modified nucleotide is "G", as shown here.
Figure 47Leek Cullinl coding sequence "modified" SEQ IDNO. 47. The nucleotide between brackets and bold indicates the position of the SNP 147 bp from the start. The modified nucleotide is "G", as shown here. Figure 48Lettuce Cullinl coding sequence "modified" SEQ IDNO. 48. The nucleotide between brackets and bold indicates the position of the SNP 141 bp from the start. The modified nucleotide is "G", as shown here. Figure 49Radish Cullinl coding sequence "modified" SEQ IDNO. 49. The nucleotide between brackets and bold indicates the position of the SNP 138 bp from the start. The modified nucleotide is "G", as shown here. Figure 50Spinach Cullinl coding sequence "modified" SEQ IDNO. 50. The nucleotide between brackets and bold indicates the position of the SNP 141 bp from the start. The modified nucleotide is "G", as shown here. Figure 51Beetroot Cullinl coding sequence "modified" SEQ IDNO. 51. The nucleotide between brackets and bold indicates the position of the SNP 141 bp from the start. The modified nucleotide is "G", as shown here. Figure 52Cucumber Cullinl protein sequence "modified" SEQ IDNO. 52. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 53Melon Cullinl protein sequence "modified" SEQ IDNO. 53. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 54Squash Cullinl protein sequence "modified" SEQ IDNO. 54. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 55Watermelon Cullinl protein sequence "modified" SEQ IDNO. 55. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 56Eggplant Cullinl protein sequence "modified" SEQ IDNO. 56. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 57Tomato Cullinl protein sequence "modified" SEQ IDNO. 57. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 58Pepper Cullinl protein sequence "modified" SEQ IDNO. 58. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here.
WO 2017/042270 PCT/EP2016/071177 15 PCT/EP2016/071177 WO 2017/042270 Figure 59Cabbage Cullinl protein sequence "modified" SEQ IDNO. 59. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 60Carrot Cullinl protein sequence "modified" SEQ IDNO. 60. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 61Celery Cullinl protein sequence "modified" SEQ IDNO. 61. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 62Cichory Cullinl protein sequence "modified" SEQ IDNO. 62. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 63Endive Cullinl protein sequence "modified" SEQ IDNO. 63. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 64Leek Cullinl protein sequence "modified" SEQ IDNO. 64. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 65Lettuce Cullinl protein sequence "modified" SEQ IDNO. 65. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 66Radish Cullinl protein sequence "modified" SEQ IDNO. 66. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 67Spinach Cullinl protein sequence "modified" SEQ IDNO. 67. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 68Beetroot Cullinl protein sequence "modified" SEQ IDNO. 68. The amino acid between brackets and bold indicates the position of the amino acid change caused by the SNP, amino acids from the start. The modified amino acid is ‘M’, as shown here. Figure 69The multiple sequence alignment of the Cullinl coding sequence orthologues (wild type) of various crops: brassica (SEQ IDNO. 8), radish (SEQ IDNO. 15), beet (SEQ IDNO. 17), spinach (SEQ IDNO. 16), leek (SEQ IDNO. 13), squash (SEQ IDNO. 3), watermelon (SEQ IDNO. 4), cucumber (SEQ IDNO. 1), melon (SEQ IDNO. 2), tomato (SEQ IDNO. 6), eggplant (SEQ IDNO. 5), pepper (SEQ IDNO. 7), lettuce (SEQ IDNO. 14), chicory (SEQ IDNO. 11), endive (SEQ IDNO. 12), carrot (SEQ IDNO. 9), celery (SEQ IDNO. 10). Between brackets and PCT/EP2016/071177 WO 2017/042270 bold is indicated the SNP on position 147 in the Cucumber coding sequence and for other crops on a position corresponding to this position.Figure 70 The multiple sequence alignment of the Cullinl amino acid orthologues (wild type) of various crops: beet (SEQ ID NO. 34), spinach (SEQ ID NO. 33), arabidopsis (SEQ ID NO. 69), brassica (SEQ ID NO. 25), radish (SEQ ID NO. 32), leek (SEQ ID NO. 30), eggplant (SEQ ID NO. 22), tomato (SEQ ID NO. 23), pepper (SEQ ID NO. 24), cucumber (SEQ ID NO. 18), melon (SEQ ID NO. 19), watermelon (SEQ ID NO. 21), squash (SEQ ID NO. 20), celery (SEQ ID NO. 27), lettuce (SEQ ID NO. 31), endive (SEQ ID NO. 29), chicory (SEQ ID NO.28), carrot (SEQ ID NO. 26). Between brackets and bold is indicated the SNP on position 49 in the Cucumber amino acid sequence and for other crops on a position corresponding to this position.
EXAMPLESEXAMPLE 1Identification of the Cullinl gene modification in Cucumis sativusA F2 crossing population made from a commercially available "high wire" cucumber variety, "Hi Lisa", was used to create a new genetic map. In total, 375 markers and 3F2 lines are used. A QTL analysis performed on these crossing populations revealed a major QTL on chromosome 6 that causes a smaller internode length and a smaller leaf surface. Sequencing of the peak marker of the QTL revealed a SNP present in the marker sequence. The particular sequence was polymorphic in the crossing population. The nucleotide sequence of the major QTL on chromosome 6 was identified by means of BLAST. The best BLAST hits for the sequence all resembled the sequence of the Cullinl gene.
EXAMPLE 2Validation of the effect of SNP in the Cullinl gene on internode length and plant leaf area.Different populations of Cucumis sativus plants, each made with different commercially available ‘high wire’ varieties, having the phenotype of shorter internodes, and smaller leaves, were phenotypically and genetically analysed. See Table 1 for the phenotypic and genetic data. Plants were measured 3 weeks after sowing. For estimating the leaf area, from the second leaf on (not the cotyledons) all leafs present were measured, and the width and the length of a leaf were measured and multiplied with each other to obtain a (roughly estimation) score for leaf area. In the third column of Table 1, the different haplotypes for the Cullinl gene SNP are given. The score A means that the SNP marker scored homozygous wildtype Cullinl gene, B means homozygous modified Cullinl gene.In the first population, plants that are homozygous for the modified Cullinl gene (B), show an internode length that is on average 63% of the length of the plants that score homozygous for the wild type Cullinl gene (A). The B plants (homozygous for the modified Cullinl gene) show a leaf area that is on average 38% of the length of the A plants (homozygous for the wild type Cullinl gene).In the second population, B plants show on average an internode length that is 78% and a leaf area that is 39% of the A plants of the same population.In the third population, the B plants show on average an internode length of 66% and a leaf area of 47%, compared to the average of the A plants.
Table 1Results of phenotypic and genotypic analyses of 3 different cucumber lines derived from commercially available high wire varieties. The internode length is defined as the length of the main stem divided by the number of internodes. The leaf area is roughly estimated by measuring from all leafs present on a plant starting with the second leaf (not the cotyledons) the length and the width, multiplying length and width, and computing the average per plant. For the scores of the Cullinl SNP, score A means that the marker scored A homozygous (wildtype), B means homozygous (modified).
WO 2017/042270 PCT/EP2016/071177 Plnnl material Cullinl I 1 aplutpe Inlernode length (11/1) Leaf area (J K> BPQ -3 B 6 210BPQ -8 B 4,7 210BPQ -10 B 5,8 289BPQ -12 B 4,8 210BPQ -5 A 6,6 462BPQ -6 A 9,3 550BPQ -4 A 7,6 575BPQ -7 A 8,4 650BPQ -1 A 8,3 725BPQ -1 1 A 8,5 676BPQ -9 A 10,3 650 PCT/EP2016/071177 WO 2017/042270 131,.3402-2 pl-l B 4,3 208131 3402-2״ pi-15 B 5,7 285131 3402-2״ pl-7 B 5,8 23813E.3402-2 pi-9 B 6,1 216I3E.3402-2 pi-17 B 6,6 238I3E.3402-2 pl-6 A 6,7 5501313402-2 ״ pi-3 A 6,8 616I3E.3402-2 pi-10 A 6,8 567I3E.3402-2 pi-13 A 7,2 675I3E.3402-2 pl-5 A 8 690I3E.3402-2 pl-8 A 8,6 546 12E. 1480-2 pl-4 B 3,9 156I2E. 1480-2 pl-8 B 4,1 1951211480-2 ״ pl-6 B 4,3 2381211480-2 ״ pl-2 B 4,4 1951211480-2 ״ pl-l 1 B 4,8 2081211480-2 ״ pl-5 B 4,9 2241211480-2 ״ pl-9 A 5,1 3361211480-2 ״ pi-10 A 7 4801211480-2 ״ pi-14 A 7,3 4371211480-2 ״ pi-12 A 7,4 483 EXAMPLE 3Creation of a melon plant with a Cullinl gene mutation; Genetic modification of plants by ethyl methane sulfonate (ems) and identification of plants which have the mutated Cullinl gene.Melon seeds were treated with EMS by submergence of approximately 5000 seeds into an aerated solution of 0.07% (w/v) EMS during 24 hours at room temperature. The treated seeds were germinated on paper in a small plastic container and the resulting plants were grown and self-pollinated in a greenhouse to produce seeds. After maturation, these seeds were harvested and bulked in one pool. The resulting pool of seeds was used as starting material to identify the individual plants that show smaller internode length and/or smaller leaf area.The Cullinl mutants which were obtained were grown in a greenhouse in order to produce lines by self-fertilisation. Melon plant lines were analysed to confirm the smaller internode

Claims (18)

1./ CLAIMS 1. Modified Cullin1 gene comprising a modification in the wild type Cullin1 nucleotide sequence of SEQ ID No: 1:, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 which leads to a change in the wild type Cullin1 amino acid sequence of SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO. 33, SEQ ID NO: 34 respectively, wherein the modification of the nucleotide sequence is a change from Adenine, Cytosine or Thymine to Guanine on position 147 of the cucumber nucleotide sequence of SEQ ID No: 1, or, for a crop other than cucumber, on a position corresponding to position 147 of the cucumber nucleotide sequence of SEQ ID No: 1, leading to an amino acid change on position 49 of the cucumber amino acid sequence of SEQ ID No: 18, or, for a crop other than cucumber, on a position corresponding to position 49 of the cucumber amino acid sequence of SEQ ID No: 18.
2. Modified Cullin1 gene as claimed in claim 1, wherein the modified Cullin1 gene, when present in a plant, results in said plant showing a compact growth phenotype as compared to an isogenic plant of the same species not comprising the modification of the Cullin 1 gene.
3. Plant, comprising the modified Cullin1 gene as claimed in claim 1 or 2.
4. Plant as claimed in claim 3, wherein the plant belongs to a species selected from the group consisting of Cucumis sativus, Cucumis melo, Curcurbita pepo, Citrullus lanatus, Solanum melongena, Solanum lycopersicum, Capsicum annuum, Brassica oleracea, Daucus carota, Apium graveolens, Cichorium intybus, Cichorium endivia, Allium ampeloprasum, Lactuca sativa, Raphanus sativus, Spinacia oleracea, and Beta vulgaris.
5. Plant as claimed in claim 3 or 4, wherein the modified Cullin1 gene results in the plant showing a compact growth phenotype as compared to an isogenic plant of the same species not comprising the modification of the Cullin 1 gene.
6. Plant seed, comprising the modified Cullin1 gene as claimed in claim 1 or 2.
7. Plant seed as claimed in claim 6, wherein the plant seed belongs to a species selected from the group consisting of Cucumis sativus, Cucumis melo, Curcurbita pepo, Citrullus lanatus, Solanum 257500/ melongena, Solanum lycopersicum, Capsicum annuum, Brassica oleracea, Daucus carota, Apium graveolens, Cichorium intybus, Cichorium endivia, Allium ampeloprasum, Lactuca sativa, Raphanus sativus, Spinacia oleracea, and Beta vulgaris.
8. Propagation material capable of developing into and/or being derived from a plant as claimed in any one of the claims 3-5, wherein the propagation material comprises the modified Cullingene as claimed in claim 1 or 2 and wherein the propagation material is selected from a microspore, pollen, ovary, ovule, embryo, embryo sac, egg cell, cutting, root, hypocotyl, cotyledon, stem, leaf, flower, anther, seed, meristematic cell, protoplast, or cell, or tissue culture thereof.
9. The modified Cullin1 gene as claimed in any one of the claims 1 or 2 for use in the development of a plant showing a compact growth phenotype as compared to an isogenic plant not comprising the modified Cullin 1 gene.
10. The modified Cullin1 gene as claimed in any one of the claims 1 or 2 or a part thereof for use in identifying a plant showing a compact growth phenotype as compared to an isogenic plant not comprising the modified Cullin 1 gene.
11. Any of the sequences of SEQ ID No: 1 – SEQ ID No: 17 and/or SEQ ID No: 35-- SEQ ID No: 51 or a part thereof, or a marker derived thereof, for use as a marker for identifying a plant showing a compact growth phenotype as compared to an isogenic plant not comprising the modified Cullin 1 gene.
12. The modified Cullin1 gene for use of claims 13 or 14 or the sequences for use of claim 15, wherein the plant belongs to a species selected from the group consisting of Cucumis sativus, Cucumis melo, Cucurbita pepo, Citrullus lanatus, Solanum melongena, Solanum lycopersicum, Capsicum annuum, Brassica oleracea, Daucus carota, Apium graveolens, Cichorium intybus, Cichorium endivia, Allium ampeloprasum, Lactuca sativa, Raphanus sativus, Spinacia oleracea, and Beta vulgaris.
13. Method for obtaining a plant which shows a compact growth phenotype comprising; a) crossing a plant comprising the modified Cullin1 gene as claimed in claim 1 or with a plant not comprising the modified Cullin1 gene, to obtain an F1 population; b) optionally performing one or more rounds of selfing and/or crossing a plant from the F1 to obtain a further generation population; c) selecting a plant that has a compact growth phenotype and the modified Cullingene as defined in claim 1 or 2. 257500/
14. Method for obtaining a plant which shows a compact growth phenotype comprising; a) crossing a plant comprising the modified Cullin1 gene of the current invention with another plant comprising the modified Cullin1 gene, to obtain an F1 population; b) optionally performing one or more rounds of selfing and/or crossing a plant from the F1 to obtain a further generation population; and c) selecting a plant that has a compact growth phenotype and the modified Cullin1 gene as defined in claim 1 or 2.
15. Method as claimed in claim 13 or 14, wherein the plant belongs to a species selected from the group consisting of Cucumis sativus, Cucumis melo, Curcurbita pepo, Citrullus lanatus, Solanum melongena, Solanum lycopersicum, Capsicum annuum, Brassica oleracea, Daucus carota, Apium graveolens, Cichorium intybus, Cichorium endivia, Allium ampeloprasum, Lactuca sativa, Raphanus sativus, Spinacia oleracea, and Beta vulgaris.
16. Marker for identifying a plant showing a compact growth phenotype, comprising the modified Cullin1 gene as claimed in claim 1 or 2 or a part thereof that comprises the modification.
17. Marker as claimed in claim 16, wherein the modification is a nucleotide substitution on or around position 147 of SEQ ID No:1 of cucumber, or, in case of a crop other than cucumber, on or around a position corresponding to position 147 of SEQ ID No:1 of cucumber, which modification leads to an amino acid substitution in the Cullin1 protein.
18. Method for selecting a plant showing a compact growth phenotype from a population of plants, comprising detecting the presence or absence of a guanine on position 147 of the cucumber nucleotide sequence of SEQ ID NO:1, or, for a crop other than cucumber, on a position corresponding to position 147 of the cucumber nucleotide sequence of SEQ ID No: 1, in the genome of a plant of a population of plants, and selecting a plant comprising a guanine on position 147 of SEQ ID NO:1, or, for a crop other than cucumber, on a position corresponding to position 147 of the cucumber nucleotide sequence of SEQ ID No: 1. For the Applicants, REINHOLD COHN AND PARTNERS
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